SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNasePossessing Three Different Activities Trypsin-like, Metalloprotease and DNaseAnna M. Timofeeva1, Valentina N. Buneva1,2 and Georgy A. Nevinsky1,2*

1Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave., Novosibirsk 630090, Russia2Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia*Corresponding author: Georgy A. Nevinsky, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, NovosibirskState University, Novosibirsk 630090, Russia, Fax: +7(383)3635153; Tel: +7(383)3635126; E-mail: nevinsky@niboch.nsc.ru

Received date: April 17, 2017; Accepted date: May 20, 2017; Published date: May 29, 2017

Copyright: ©2017 Timofeeva AM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Objective: DNA- and myelin basic protein (MBP)-hydrolyzing antibodies can play an important role in thepathogenesis of systemic lupus erythematosus (SLE), therefore their analysis seems to be important.

Results: In mammalians there are serine proteases, metalloproteases, and DNases. Each of these and manyother enzymes catalyze only one chemical reaction. Very unusual and unpredictable situation was revealed by us inthe case of monoclonal abzymes corresponding to the sera of patients with SLE. The small pools of phage particlesdisplaying light chains with different affinity for myelin basic protein (MBP) were isolated by affinity chromatographyon MBP-Sepharose. In contrast to canonical enzymes, one of twenty five MLChs demonstrated three differentenzymatic activities; it efficiently hydrolyzed MBP (but not other proteins) and DNA. Other twenty four MLChshydrolyzed only MBP. The proteolytic activity of NGTA3-pro-DNase was efficiently inhibited by specific inhibitors ofserine-like (PMSF) and metalloproteases (EDTA). Protease and DNase properties of NGTA3-pro-DNase differsignificantly from those for the corresponding canonical enzymes.

Conclusion: This is the first example of monoclonal antibodies with three different catalytic activities. Theprincipal possibility of the existence of monoclonal antibodies with several different enzymatic activities isunexpected but very important for the further understanding of unknown biological functions of humanimmunoglobulins.

Keywords: Systemic lupus erythematosus; Recombinant monoclonallight chain; Hydrolysis of myelin basic protein and DNA

Abbreviations: Ab: Antibody; EDTA: Ethylenediamine TetraaceticAcid; HAS: Human Serum Albumin; IPTG: Isopropyl β-D-1-Thiogalactopyranoside; MBP: Myelin Basic Protein; OP: Oligo Peptide;PFU: Plaque-Forming Unit; SDS-PAGE: SDS-Polyacrylamide GelElectrophoresis; MLCh: Monoclonal Light Chain; MS: MultipleSclerosis; SLE: Systemic Lupus Erythematosus; RA: Relative Activity.

IntroductionAbzymes against transition chemical states of different reactions

were studied intensively reviewed in [1-3]. Similarly to artificialabzymes against analogs of transition states of catalytic reactions [1-3],naturally occurring abzymes may be antibodies (Abs) raised directlyagainst enzyme substrates acting as haptens and mimicking transitionstates of catalytic reactions [3-6] or may be antiidiotypic Abs againstactive centers of various enzymes [7,8].

A special feature of autoimmune diseases including systemic lupuserythematosus (SLE) and multiple sclerosis (MS) is highconcentrations of auto-Abs to many different endogenous antigensincluding DNA and myelin basic protein (MBP) [3-6]. Polyclonalnatural IgG and/or IgA and IgM abzymes hydrolyzing DNA, RNA,polysaccharides, nucleotides, oligopeptides, and proteins from the seraof patients with several autoimmune and viral diseases were revealed

(reviewed in [3-6]). Healthy humans and patients with many diseaseswith insignificant autoimmune reactions usually lack abzymes ordevelop Abs with very low catalytic activities, often on a borderline ofthe sensitivity of detection methods.

It was shown that electrophoretically homogeneous polyclonal Absfrom the sera of MS [9-13] and SLE [14-17] patients contains smallfractions with DNase and MBP-hydrolyzing activities, which areintrinsic properties of IgGs and/or IgMs and IgAs. SLE and MSpatients can generate polyclonal abzymes attacking MBP in themyelin-proteolipid sheath of axons and DNA and can play animportant role in these diseases pathogenesis [6]. There are examplesof monoclonal human [18,19] and mouse IgGs hydrolyzing DNA[20,21]. Bence-Jones proteins from patients with multiple myelomashould be considered as the first found natural human monoclonalabzymes [22]. Monoclonal light chain (MLCh) of anti-VIP abzyme wasexpressed in bacteria, purified, and found to possess an intrinsic VIP-hydrolyzing activity [23,24]. Two MLChs from patients with multiplemyeloma with prothrombinase activity were identified [25]. TwoMLChs from multiple myeloma patients specifically hydrolyzed viralgp120 protein [26,27]. Recently MLChs capable to degrade the activesite of the urease of Helicobacter pylori and eradicate the bacterialinfection in a mouse stomach was obtained [28]. Catalytic MLCh wasobtained by immunizing with a peptide possessing a part of a sequenceof a chemokine receptor, CCR-5, which is present as a membraneprotein on the macrophage surface [29].

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It should be mentioned, that all described above monoclonalabzymes catalyzed only one chemical reaction. At the same time, theorigin of classical enzymes and catalytic antibodies has a completelydifferent nature. To each canonical enzyme corresponds one gene thatencodes its protein structure. The formation of genes encodingstructure of Abs occurs due to V(D)J recombination, which is theunique mechanism of genetic recombination that occurs only indeveloping lymphocytes during the early stages of T and B cellmaturation [30]. The process results in the highly diverse repertoire ofimmunoglobulins and it is a defining feature of the adaptive immunesystem and its development was a key event in the evolution of jawedvertebrates. The process ultimately results in novel amino acidsequences in the antigen-binding regions of immunoglobulins thatallow for the recognition of antigens from nearly all pathogens; therecognition can be also "autoreactive" and leading to autoimmunity[30]. It should be mentioned, that immunization of autoimmune miceresults in a dramatically higher incidence of abzymes with a higheractivity than in conventionally used normal mouse strains [31,32],therefore the formation of abzymes in autoimmune diseases may bemuch more profuse. In addition, theoretically the immune system canproduce 106 different Ab variants in response to a single antigen [33].Moreover, an increased level of apoptosis in autoimmune patients maytrigger production of abzymes directly to DNA and its complexes withvarious proteins. Therefore, situation with catalytic antibodies may bevery complicated and somewhat unpredictable.

For example, DNases hydrolyze only DNA and are inactive in thehydrolysis of RNA, while RNases cannot hydrolyze DNA. Severalmonoclonal mouse SLE IgGs against B-DNA of different sequencesefficiently hydrolyze single- and double-stranded DNA and RNA in asequence-independent manner, with the hydrolysis of RNA being 30–100-fold faster than of DNA [20]. In addition, immunization of rabbitswith DNA, RNA, DNase I, DNase II, and pancreatic RNase Aproduced IgGs with intrinsic DNase and RNase activities ([34] andreferences therein). It was shown that small fractions of thepreparations of total polyclonal IgGs against DNase I, DNase II, andRNase demonstrate high DNase and RNase activities belong toantiidiotypic Abs to these enzymes, but they also contain smallfractions interacting no with Sepharose bearing Abs against theseenzymes, but interact with nucleic acids bound to these enzymes [34].It means that these Abs are against nucleic acids bound with theenzymes used.

An immunoglobulin light chain phagemid library derived fromperipheral blood lymphocytes of patients with SLE was recently used[35]. Small pools of phage particles displaying light chains withdifferent affinity for MBP were isolated by affinity chromatography onMBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used forpreparation of individual monoclonal light chains (MLChs, 27-28kDa). Seventy five of 440 individual colonies were randomly chosen,expressed in E. coli in a soluble form, and MLChs were purified bymetal chelating chromatography. Twenty-five of 72 MLChs efficientlyhydrolyzed MBP. Four of twenty-two MLChs analyzed in this article[35] demonstrated serine protease-like and three, thiol protease-likeactivities, while eleven MLChs were metalloproteases. The activity ofthree chimeric MLChs was inhibited by both PMSF and EDTA, twoother by EDTA and iodoacetamide and one by PMSF, EDTA, andiodoacetamide [35]. These observations suggest an extreme diversity ofanti-MBP abzymes in SLE patients.

Two of 25 MLChs were later analyzed in details [36,37].Uninspected results were obtained. The activity of one MLCh-23

(NGTA1-Me-pro) was inhibited only by EDTA and it efficientlyhydrolyzed MBP (but not other control proteins) [36]. NGTA1-Me-prowas shown to be abzyme with two independent metalloprotease activecenters demonstrating two pH optima, two optimal concentrations ofMe2+ ions, and two Km values for MBP [36].

MLCh-24 (NGTA2-Me-pro-ChTr) also efficiently hydrolyzed onlyMBP [37]. Its proteolytic activity was efficiently inhibited only byspecific inhibitors of serine-like (PMSF) and metalloproteases (EDTA).It was shown, that MLCh-24 possesses independent serine-like andmetal-dependent activities [37].

We report in this article first example of even more unusualrecombinant MLCh-25, which binds and hydrolyzes DNA, MBP, andthree oligopeptides corresponding to its four specific sequences(immunodominant sequences containing cleavage sites)demonstrating three different alternative activities: two proteolytic(serine-like and metal-dependent) and DNase activity.

Methods

Materials and chemicalsMost chemicals, proteins and the Superdex 200 HR 10/30 column

were from Sigma, while chelating Sepharose from GE Healthcare.Human MBP was from the Department of Biotechnology, ResearchCenter of Molecular Diagnostics and Therapy (Moscow). MBP-Sepharose was obtained by immobilizing of MBP on BrCN-activatedSepharose according to the standard manufacturer’s protocol.

Amplification of phage libraryWe have used human lupus kappa light chains library (from three

patients; 106 variants of different light chains); cDNA was cloned intothe phagemid pCANTAB5His6 vector after a leader sequence of thephage coat protein gene pIII of a filamentous E. coli bacteriophageM13 between SflI and NotI restriction sites using standard methods[24,38,39]. This library was a gift from S. Paul and S. Plaque(University of Texas Houston Medical School, USA); all details of thislibrary preparation were described earlier [24]. Amplification of theVCSM13 helper phage and determination of its titer was carried outaccording to [24]. The amplification of the phage library was carriedout as in [24,38,39].

Chromatography of phage particles on MBP-SepharosePreparations prepared using E. coli TG1 were exhausted from the

main part of phage particles having affinity to DNA bychromatography on DNA-cellulose according to [18,19]. Thenpreparations of phage particles eluted from DNA-cellulose at loading(4 ml containing 2.5 × 1012 phage particles) were applied onto MBP-Sepharose column (3 ml) equilibrated with 20 mM Tris-HCl (pH 7.5)and the column was washed with the same buffer to zero opticaldensity (A280). For the control similar solution of phage particlescorresponding to pCANTAB plasmid containing no library of lightchains was used. The phage particles were eluted with the same buffercontaining different concentrations of NaCl (0.01–3 M), and then with50 mM glycine-HCl (pH 2.6) similarly to purification of polyclonalAbs [16,17]. Phage particles were collected, concentrated, and eachfraction was precipitated using PEG/NaCl as in [24]. The titers ofphage particles were determined (see below) and each fraction wasassayed for MBP-hydrolyzing activity using intact MBP and four

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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oligopeptides (OPs) corresponding to the protein specificimmunodominant sequences containing cleavage sites (see below).

Preparation of monoclonal phage particlesFor preparation of soluble MLChs we have used E. coli HB2151 as

in [35-37]. An overnight culture of E. coli HB2151 (200 μl) was placedin a flask containing 80 ml of 2YTL medium and this mixture wasincubated with shaking at 37°C to A600=1.0. The cells were centrifugedfor 10 min (4000 rpm), and the pellet was re-suspended in 80 ml of 10mM MgSO4 for receiving a solution containing cells in initialconcentration. The preparation of phage particles eluted from MBP-Sepharose with 0.5 М NaCl was diluted 100-fold in 2YTL, and 10 μl ofthis solution was added to 90 μl of the E. coli cells. The mixture wasincubated for 30 min at 37°C and uniformly distributed over a Petridish with agarized 2YTL containing 40 μg/ml ampicillin; the dish wasincubated overnight at 37°C. For further analysis, 72 of 440 individualcolonies from two dishes were randomly chosen.

To propagate individual colonies of phages, the material from eachcolony were grown overnight at 37°C in a Petri dish as describedabove. The cells were scraped to a vial containing 1.5 ml 2YTL mediumsupplemented with ampicillin (50 μg/ml), and the mixture was shakenat 37°C to A600=0.6. Then 1.5 ml of 2YTL and isopropyl β-D-1-thiogalactopyranoside (IPTG) to the final concentration 2 mM wereadded, and the mixture was shaken at 37°C overnight. The suspensionwas centrifuged (1 min, 12000 rpm); the supernatant containing phageparticles was collected and used to obtain monoclonal phages.

Purification of monoclonal light chainsSupernatant (50 ml) containing MLCh was dialyzed twice for 3 h at

4°C against 1 ml of H2O and then overnight against Buffer Aconsisting of 50 mM Tris-HCl (pH 7.2), 0.5 M NaCl, and 1 mM DTT.The solution obtained was first applied on column with Sephadex G-75for removal of different hydrophobic compounds and then on a HiTrapchelating Sepharose column (1 ml) charged with Ni2+ and equilibratedin buffer A for affinity chromatography [35-37]. After loading MLCh,the column was washed with the same buffer to zero optical density ofthe eluate. The bound MLChs were eluted with a gradient of imidazole(0-1 M) in 50 mM Tris-HCl (pH 7.2). Optical density was measured inall fractions. The fractions containing MLChs were dialyzed against 20mM Tris-HCl (pH 7.5) and then concentrated. According of SDS-PAGE analysis these preparations contain small admixtures of severalproteins (probably from medium, E. coli cells or phage particles)interacting with HiTrap chelating Sepharose. For isolation ofhomogeneous preparations of MLCh we used gel filtration. FPLC gelfiltration of this preparation was performed on a Superdex 200 HR10/30 column equilibrated with 50 mM Tris-HCl (pH 7.5) containing0.3 M NaCl as in [16,17,35-37]. Before gel filtration, the MLCh sampleswere incubated in TBS containing 2.0 M MgCl2 for 30 min at 20°C.The fractions containing [26-27] kDa MLCh were collected. About0.3-0.8 mg of MLChs was obtained from 1 liter of the medium,depending on the preparation. Only fraction corresponding to theMLChs (containing no proteins admixtures) demonstrated MBP-hydrolyzing activity.

In order to protect the MLChs from bacterial contamination theywere sterilized by filtration through a Millex filter (pore size 0.2 μm)and then concentrated in sterile condition and used for analysis.Incubation of standard bacterial medium with MLChs preparationsdid not lead to a formation of colonies. In this article we have analyzed

in detail only one MLCh (NGTA3-pro-DNase) demonstrating highMBP- and DNA-hydrolyzing activities (see above).

Western blotting and ELISAThe NGTA3-pro-DNase was analyzed by Western blotting onto a

nitrocellulose membrane using horseradish peroxidase conjugatedwith mouse Abs against light chains of human Abs as in [16,17,35-37].The interaction of the MLChs with MBP was also analyzed usingstandard ELISA plates with immobilized MBP [16,17,35-37], ordouble-stranded DNA [18,19]. After adsorption of the MLChs and aconsecutive treatment of samples with horseradish peroxidaseconjugated with mouse antibodies against light chains of human Absthe reaction mixtures were incubated with tetraethyl benzidine andH2O2. The reaction was stopped with H2SO4 and optical density (A450)was determined. The relative content of anti-MBP and anti-DNAMLChs in the samples was expressed as a difference in the relativeabsorption at 450 nm between experimental and control samples;controls with MBP (or DNA) but without MLChs were used. Controlswith DNA but without MLChs as well as with MLChs not interactingwith DNA gave the same results.

Proteolytic activity assayThe reaction mixture (10-40 μl) for analysis of MBP- or

oligopeptide-hydrolyzing activity of MLCh, containing 20 mM Tris-HCl (pH 7.5), 0.5-1.0 mg/ml (28-56 μM) MBP or 0.33-1.0 mM one offour different oligopeptides, and 0.001-0.01 mg/ml (37-370 nM) ofMLCh, was incubated for 0.1-24 h at 30°C. OP-17 (X-ENPVVHFFKNIVTPRTP), OP-19 (X-LSRFSWGAEGQLPGFGYGG),OP-21 (X-YLASASTMDHARHGFLPRRHR), and OP-25 (X-AQGTLSKIFKLGGRDSRSGSPMARR) oligopeptides corresponding tofour known IgG-dependent specific cleavage sites of MBP [13,16,17]and containing fluorescent residue 6-O-(Carboxymethyl) fluoresceinethyl ester (R) on its N-terminus were used.

The MBP cleavage products were analyzed by SDS-PAGE in 12% or4-15% gradient gels with Coomassie R250 staining. The gels wereimaged by scanning and quantified using GelPro v3.1 software. Finally,the activities of the MLCh preparations were determined as a decreasein the percentage of MBP converted from the initial to hydrolyzedforms taking into account incubation of MBP in the absence of Abs.

The cleavage products of different OPs were separated by TLC onKieselgel F60 plates using acetic acid-n-butanol-H2O (1:4:5) system.The plates were dried and photographed. To quantify the intensities ofthe fluorescent spots after TLC, OPs incubated without MLCh wereused as controls. Photographs of the plates were imaged by scanningand quantified using GelPro v3.1 software. All quantitativemeasurements (initial rates) were taken under the conditions of thepseudo-first order of the reaction within the linear regions of the timecourses (15-40% of MBP or hydrolysis of OPs) and dependence of therate on MLCh concentration.

pH dependencies were analyzed using different buffers (50 mM):MES-NaOH (pH 5.4-6.6), Tris-HCl (pH 6.0-8.6) and glycine-NaOH(pH 9.0-10.0). In some cases, MgCl2, MnCl2, CuCl2, CoCl2, NiCl2,ZnCl2, or CaCl2 at different concentrations were used. For analysis of apossible type of proteolytic activity, MLCh (0.3-0.5 mg/ml) was pre-incubated for 30 min at 25°C with one of specific inhibitors of differentproteases: iodoacetamide (10 mM), PMSF (1 mM), or EDTA (10-100mM), and aliquots of these mixtures were then added to the standardreaction mixture. Substrate specificity of NGTA3- pro-DNase was

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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analyzed using MBP, human serum albumin, human milk lactoferrinand four different OPs.

DNase activity assayDNase activity was analyzed using supercoiled (sc) DNA as

described earlier for DNase I, DNase II, and human serum catalyticantibodies [9,10]. Initially, the reaction mixtures (20 μl) contained 18μg/ml supercoiled pBluescript DNA, 5 mM MgCl2, 1 mM EDTA, 20mM Tris-HCl (pH 7.5), 2 μl of the phage preparation afterchromatography on DNA-cellulose or purified light chain NGTA3-pro-DNase (1-10 nM), and were incubated for 0.5-2 h at 37°C.

Later, the specific optimal conditions were used. Dependencies ofthe relative DNase activity on the concentration of metal ions wereanalyzed using KCl, NaCl, MnCl2, MgCl2, CaCl2, CuCl2, CoCl2, NiCl2,or ZnCl2 (each at 0.005-20 mM).

After incubation 3 μl of buffer containing 1% SDS, 20 mM EDTA,pH 8.0, 30% glycerol and 0.005% bromophenol blue was added toreaction mixture. Electrophoresis was performed in 0.8% agarose geluntil the bromophenol blue migration to 2/3 of the way. DNA gelstained with ethidium bromide (0.5 μg/ml, 2-4 h). The images ofethidium bromide-stained gels were captured on a Sony DSC-F717camera and a relative amount of DNA in different bands was analyzedusing ImageQuant v5.2 (Molecular Dynamics, GE Healtcare, UK).

Similarly to DNase I and polyclonal abzymes from autoimmunepatients [9,10,40,41], depending on the incubation time and type ofmetal ions, MLCh was capable to hydrolyze plasmid DNA formingsingle breaks in one strand of supercoiled DNA (relaxed DNA) andthen multiple breaks yielding linear DNA. Finally, it hydrolyzed DNAinto short and medium-length oligonucleotides.

However, after such a deep hydrolysis of DNA, it was very difficultto estimate relative activity of MLCh. Therefore, to estimate the DNaseactivity quantitatively, we have found the concentration of MLChpreparation and the time of incubation sufficient to convert scDNAinto the relaxed form without further noticeable fragmentation after0.2-2 h of incubation. Finally, the activities of the MLCh preparationswere determined as a decrease in the percentage of DNA convertedfrom the initial supercoiled form to the relaxed form (and sometimesadditionally linear form), corrected for the distribution of DNAbetween these bands in the control (incubation of pBluescript in theabsence of Abs).

All measurements (initial rates) were taken within the linear regionsof the time courses (15-40% of DNA hydrolysis) and a completetransition of the supercoiled plasmid (18 µg/ml=6.0 nM) to the nickedform was taken for 100% activity. This approach allows normalizationof the relative activity of MLChs and calculation of kcat value.

In some experiments, the MLChs were extensively dialyzed against50 mM Tris-HCl (pH 7.5) containing 0.1 M EDTA and then threetimes against 1 mM Tris-HCl (pH 7.5); before the experiments, metalions were removed from all solutions using a 5 ml Chelex column. TheRAs of these preparations were assayed as described above using astandard reaction mixture.

Dependency of the activity on pH was analyzed using differentbuffer systems as described above for analysis of protease activity.

In gel assay of protease activityAnalysis of MBP- and DNA-hydrolyzing activity of NGTA3-pro-

DNase after SDS-PAGE was performed similarly to [16-19,35-37].MLCh (10-15 μg) was pre-incubated at 30°C for 30 min undernonreducing condition (50 mM Tris-HCl, pH 7.5, 1% SDS, and 10%glycerol). After standard SDS-PAGE electrophoresis of MLCh torestore its MBP-hydrolyzing activity, SDS was removed by incubationof the gel for 1 h at 30°C with 4 M urea and washed 10 times (7-10min) with H2O.

Then 2-3-mm cross sections of longitudinal slices of the gel were cutup and incubated with 50 μl of 50 mM Tris-HCl, pH 7.5, containing 50mM NaCl for 4-6 days at 4°C to allow protein refolding and elutingfrom the gel. The solutions were removed from the gels bycentrifugation and used for assay of MBP and DNA hydrolysis asdescribed above. Parallel control longitudinal lanes were used fordetecting the position of MLCh on the gel by Coomassie R250staining.

In situ analysis of DNase activityDNase activity of NGTA3-pro-DNase was determined in situ after

separation of proteins in 4-15% gradient SDS-PAGE gels containing 3μg/ml calf thymus DNA as in [18,19,42].

For the analysis of DNase activity (and to remove SDS), the gelswere washed at 25°C for 4 h in 40 mM Tris-HCl, pH 7.5, containing0.1% Triton X-100, 4 mM MgCl2, and 0.2 mM CaCl2 and thenincubated in the same buffer containing 0.1 mM CaCl2 but withoutTriton X-100 for 2 h followed with a fresh portion of the same bufferfor 1-2 days at 30°C. To visualize the regions of DNA hydrolysis, thegels were stained with ethidium bromide. The parallel longitudinalslices were used to detect the position of the MLCh in the gel byCoomassie R250 staining.

Fluorescence measurementsFluorescence was measured in a thermostated (25°C) Hitachi

MPF-2A spectrofluorimeter. Excitation was performed at 296 nm andfluorescence emission detected at 330 nm. The complex formationmixture contained 20 mM Tris-HCl (pH 7.5), and 0.3 mg/ml MBP.Aliquots (0.2-0.5 μl) of d (TAGAAGATCAAA) oligonucleotide wereconsecutively added to the mixture, and changes in MBP fluorescencespectrum (∆F) were recorded, with correction for dilution.

The Kd values of MBP: oligonucleotide complexes were calculatedfrom the Scatchard equation ∆F=∆Fmax–Kd(∆F/[L]), where [L] is theconcentration of free oligonucleotide in the mixture [43]. Theestimation error did not exceed 5-10%.

Determination of kinetic parametersThe Km and Vmax (kcat) values were calculated from the

dependencies of V versus [MBP] or [DNA] by least-squares non-linearfitting using Microcal Origin v5.0 software and presented as lineartransformations using a Lineweaver-Burk plot [43]. The concentrationsof MBP and DNA were varied. Errors in the values determination werewithin 8-12%. The results are reported as mean ± S.E. of at least 2-3independent experiments.

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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Figure 1: Affinity chromatography of phage particle preparation onMBP-Sepharose: (—) and (---), absorbance at 280 nm of thematerial corresponding to phage particles with and without kappalight chains cDNA, respectively (A). Concentrations of NaCl usedand relative titers of phage particles corresponding to differentpeaks are shown. The bars indicate the relative activity (RA) of 10small pools of phage particles corresponding to peaks 1-10 elutedfrom the sorbent with different concentrations of NaCl and anacidic buffer (pH 2.6) in the hydrolysis of MBP (B) and in fourdifferent MBP oligopeptides (C); the reaction mixtures containingMBP (0.7 mg/ml) or 1 mM OPs and 5 × 107 or 108 PFUsrespectively were incubated for 6 h at 30°C. For details, seeMaterials and methods.

Figure 2: SDS-PAGE analysis of MBP- and DNA-hydrolyzingactivities (A) and homogeneity of MLCh (7 μg) using a reducing 5–16% gradient gel followed by silver staining (B, lane 1); the arrows(B, lane 2) indicate the positions of molecular mass markers. Afterelectrophoresis the gel was incubated under special conditions forrenaturation of MLCh. The relative MBP- and DNA-hydrolyzingactivity (%) was revealed using the extracts of 2-3-mm manyfragments of one longitudinal slice of the gel (A). The activity ofMLCh corresponding to a complete hydrolysis of 0.5 mg/ml MBP(or 18 nM scDNA) after 24 h of incubation of 25 μl reactionmixture containing 10 μl of the gel extracts was taken for 100%. Theaverage error in the initial rate determination did not exceed 7-10%. SDS-PAGE analysis of hydrolysis of MBP (0.5 mg/ml) incubatedfor 12 h alone (lane 1), with 0.1 mg/ml inact-MLChmix (lane 2) orfor 6 h with 0.01 NGTA3-pro-DNase mg/ml (lane 3) (C).Hydrolysis of control proteins (0.5 mg/ml) by inact-MLChmix (0.1mg/ml) and NGTA3-pro-DNase (0.1 mg/ml) was analyzed: humanserum albumin (lanes 4 and 5) and human milk lactoferrin (lanes 6and 7) (C). The mixtures were incubated for 6 h with inact-MLChmix (lanes 4 and 6), or NGTA3-pro-DNase (lanes 5 and 7).Lanes C correspond to different proteins incubated alone withoutMLChs, while lane C1- to a mixture of standard protein markerswith known molecular masses. Relative activity of NGTA3-pro-DNase in the hydrolysis of OP19, OP21, and OP25 (D). Lanes 1-3correspond to 1 mM OPs incubated alone (1), in the presence of 0.1mg/ml inact-MLChmix (2), and 0.01-0.03 mg/ml NGTA3-pro-DNase (3) for 6 h.

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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Results

Affinity chromatography of phage particles on MBP-Sepharose

Polyclonal anti-MBP Abs from the sera of SLE and MS patients, areusually very heterogeneous in their affinity for MBP and can beseparated into many subfractions by chromatography on MBP-Sepharose [16,17]. Therefore, we first separated phage particles (E.coliTG1) containing a pool of various MLChs with different affinity forMBP by chromatography on MBP-Sepharose (Figure 1A). Thecomplete pool of phage particles containing MLChs bound with MBP-Sepharose were distributed between ten peaks eluted duringchromatography (Figure 1A) and all fractions corresponding to newsmall pools obtained were active in the hydrolysis of MBP (Figure 1B)and its four OPs (Figure 1C). However, we have not observed anydetectable protein peaks having remarkable affinity for MBP-Sepharoseafter similar affinity chromatography of phage particles correspondingto pCANTAB plasmid containing no cDNA of light chains (Figure1A). It means that the pools of MLChs of all 10 fractions of phageparticles with different affinity to MBP contain not only inactive butalso catalytically active light chains possessing MBP-hydrolyzingactivity.

Preparation of individual homogeneous recombinant MLChsIndividual colonies corresponding to recombinant MLChs have

been obtained using E. coli HB2151 and phage particles eluted fromMBP-Sepharose with 0.5 M NaCl (peak 7, Figure 1A); this fractiondemonstrated relatively high affinity and catalytic activity in hydrolysisof intact MBP (Figure 1B) and its four different OPs (Figure 1C). Thephage particles of this fraction were growing on two Petri dishes withagar and separated colonies were used for preparation of individualMLChs.

On the end of recombinant MLChs there is a sequence of sixhistidine residues; this hexapeptide interacts efficiently with Ni2+ ions[24]. 72 preparations of monoclonal MLChs corresponding torandomly chosen colonies were purified by chromatography onHiTrapTM chelating Sepharose charged with Ni2+ ions and by followinggel filtration. Twenty five of 72 recombinant MLChs efficientlyhydrolyzed MBP. Twenty-four MLChs were described earlier [35-37].One recombinant NGTA3-pro-DNase monoclonal light chain (belowmarked as MLCh-25) demonstrating relatively high MBP-hydrolyzingactivity and MLChs of 5 single colonies without activity were used inthis article for more detail analysis.

The electrophoretical homogeneity of ~26-27-kDa NGTA3-pro-DNase and control preparation of equimolar mixture of 5 MLChswithout activity (inact-MLChmix) was confirmed by SDS-PAGE withsilver staining (for example, Figure 2B, lane 1).

Characterization of individual homogeneous recombinantMLChs

It was shown, that MLCh-25 and inact-MLChmix demonstratedpositive answer with mouse IgGs (conjugated with horseradishperoxidase) against human Abs light chains at Western blotting andpositive ELISA answer using plates with immobilized MBP. NGTA3-pro-DNase was active in the hydrolysis of MBP (Figure 2C) in theabsence of external metal ions (lane 2) and in the presence of CaCl2(lane 3), while inact-MLChmix preparation did not hydrolyze MBP

(Figure 2C, lane 1). To exclude possible artifacts due to hypotheticaltraces of contaminating proteases, the MLCh-25 was subjected to SDS-PAGE and its MBP-hydrolyzing activity was detected after extractionof proteins from the separated gel slices (Figure 2A).

In the absence of external metal ions and in the presence of CaCl2MBP-hydrolyzing activity was detected only in the band correspondingto the light chains (Figure 2A). Since SDS dissociates any proteincomplexes, the detection of proteolytic activity in the gel regioncorresponding only to the MLCh-25, together with the absence of anyother bands of the activity or protein (Figure 2), as well as the absencethese activities in the case of inact-MLChmix (Figures 2C and 2D)provides direct evidence that recombinant MLCh-25 possess MBP-hydrolyzing activity.

It was shown previously, that polyclonal SLE and MS IgGs purifiedon MBP-Sepharose hydrolyze only MBP, but not many other testedproteins [11,12,16,17]. Similar situation was observed in the case ofabzymes against other proteins and peptides: HIV-1 reversetranscriptase and integrase, human serum albumin, casein,thyroglobulin, and intestinal vasoactive peptide [44-47]. It has beenshown, that NGTA3-pro-DNase efficiently hydrolyzes MBP in theabsence of external metal ions (Figure 2C, lane 2) and in the presenceof 2 mM CaCl2 (Figure 2C, lane 3).

In the same conditions (in contrast to canonical proteaseshydrolyzing all proteins) there was no observed detectable hydrolysisof control non-specific proteins, (Figure 2C, lanes 4-7). In contrast toinact-MLChmix, NGTA3-pro-DNase efficiently hydrolyzed three OPscorresponding to MBP antigenic determinants (OP19, OP21 andOP25) (Figure 2D). At the same time, in contrast to canonicalproteases hydrolyzing all peptides, NGTA3-pro-DNase did notefficiently hydrolyze OP17.

Type of proteolytic activity of recombinant MLChIt was previously shown that, in contrast to polyclonal MS IgGs,

MBP-hydrolyzing abzymes from SLE patients are more sensitive toEDTA and less sensitive to PMSF, which is a specific inhibitor ofserine-like proteases [16]. Figure 3A demonstrated that NGTA3-pro-DNase is not sensitive to specific inhibitors of thiol-like(iodoacetamide) and acidic-like (pepstatin A) proteases. Preincubationof MLCh-25 containing intrinsic metal ions with specific inhibitor ofserine-like proteases (PMSF) leads to decrease in its activity for 67 ±5%. Human and mammalian intact polyclonal Abs are known tointeract with different metal ions and they do not completely loseintrinsically bound metal ions during the standard procedure of theirpurification [48,49]. The dialysis of MLCh-25 against EDTA oraddition of EDTA to MLCh containing only intrinsically bound Me2+-ions led to a decrease in its activity for 33 ± 3% (Figure 3A). Andaverage Me2+-dependent proteolytic activity of MLCh-25 containingonly intrinsically bound Me2+-ions was approximately 2.0-fold lower(Figure 2A), but after addition of external Ca2+ ions became to be 2.2-fold higher than its serine like activity (Figure 3B).

Dependence of MLCh protease activity upon different metalions

We have compared the effect of different metal ions on theproteolytic activity of MLCh-25 in the hydrolysis of MBP (Figure 3B).It was shown that in contrast to canonical metal-independent serine-,thiol-, and acidic-like proteases, seven different external metal ionsactivate NGTA3-pro-DNase in the following order: Ca2+ ≥ Ni2+>Co2+

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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≈ Mn2 ≥ Cu2+ ≈ Zn2+ ≥ Mg2+ (Figure 3B). An optimal concentration ofCaCl2, which is the best activator of MLCh-25, was 3 mM (Figure 3C).The use of the combinations of Ca2+ with other metal ions led to asignificant decrease in the metal-dependent proteolytic activity ofMLCh as compared with CaCl2 taken separately (Figure 3D).

Figure 3: The relative MBP-hydrolyzing activity of NGTA1-Me-pro(RA) after its preincubation with specific inhibitors of proteases ofdifferent types (A). MLCh (0.1 mg/ml) was preincubated in theabsence of other components (control), in the presence of PMSF,iodoacetamide, pepstatin, or EDTA, and then 1.5 μl added to 28.5 μlof standard reaction mixture containing MBP (A). The RA of MLChbefore its preincubation with different inhibitors was taken for 100%. Effect of EDTA and different metal ions (2 mM) on the RA ofMLCh is shown (B). Dependence of RA of MLCh on theconcentration of CaCl2 (C). Effect of different metal ions on the RAof MLCh in the presence of CaCl2 in optimal concentration (3 mM)(D). Dependence of the relative MBP-hydrolyzing activity of MLChon pH of reaction mixture before and after its treatment with PMSFand EDTA is given (E). The relative activity of MLCh in thehydrolysis of four different 1 mM OPs (F). For details, see Materialsand Methods.

Many known canonical human metalloproteases are calcium-dependent cysteine proteases [50,51] or Zn2+-dependent enzymes [52],while their activity in the presence of other metal ions is absent orsignificantly lower. MLCh-25 is not cysteine protease demonstratingmaximal activity in the presence of Ni2+ and Ca2+, but effects of fiveother metal ions on its activity are to some extent comparable (Figure3B). In addition, serine-like activity of MLCh-25 is approximately 2.2-fold lower that its Me-dependent activity in the presence of metal ions.

pH optimum MLCh proteolytic activityIn contrast to all human canonical proteases having one

pronounced pH optimum, polyclonal catalytic IgGs from the sera ofindividual MS and SLE patients demonstrate in the MBP hydrolysisquite distinct pH dependencies (from one to four-five pH optima)within a wide range of pH values (5-10) [11,12,16]. Taking intoaccount inhibition of MLCh-25 by PMSF and EDTA it was reasonableto expect a possibility of the existence of two optimal pH values. MLChdemonstrates two optimal pHs (Figure 3E). After treatment ofMLCh-25 with PMSF, its metalloprotease activity was maximal at pH8.6 (Figure 3E). In the presence of EDTA, serine-like protease activitydemonstrated pH optimum at 7.0 (Figure 3E). The existence of twooptimal pHs might be a consequence of accidental agglomeration oftwo different clones on Petri dishes. To obtain repeatedly chosen singlecolonies of phages, the phage material corresponding to this clone wasre-grown in Petri dishes and five new single clones were randomlychosen. Purified MLCh preparations corresponding to five new singlemono-colonies demonstrated the same two values of pH optima. Itmeans that NGTA3-pro-DNase was single after the first step of itsselection and that this recombinant MLCh-25 is characterized by twodifferent pH optima in the hydrolysis of MBP and possess serine-likeand metalloprotease activities.

Figure 4: Position of four different sequences corresponding to fourOPs in a complete protein sequence of MBP (A). Data of homologyanalysis between the sequences of OP-17, OP-19, OP-21 and OP-25is shown (B). The amino acids identical between two pairs of OPsare marked with an asterisk (*), while non-identical amino acidswith highly conserved physicochemical properties are marked witha colon (:), and those with moderately conserved properties. with adot (•).

Protein sequence site specificity of MLChSeveral abzyme-dependent specific sites of intact MBP cleavage are

clustered within four known immunodominant regions of MBP[16,17]. It was obviously, that NGTA3-pro-DNase corresponds to Absagainst one of four known immunodominant regions containingcleavage sites of MBP. Therefore, it is reasonable to assume, thatMLCh-25 will bind and hydrolyze only one of four OPs. However, anunexpected result was obtained. MLCh-25 was to some extent

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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unspecific and efficiently hydrolyzed three of four MBP oligopeptides;the activity decreased in the following order: OP21>OP19>OP25,while hydrolysis of OP17 was very insignificant (Figure 3F).

Taking into account the absence of an exceptional specificity ofMLCh in the hydrolysis of OPs, it was interesting to analyze a possiblehomology of four OPs corresponding to four different cleavage sitesdisposed in different parts of MBP (Figure 4A). Figure 4Bdemonstrates the sequence alignment of four OPs. The similaritybetween four sequences is not absolute and only some positions fullycoincide (marked with an asterisk), while several positions show good(marked with a colon) or moderate (marked with a dot) conservationof physicochemical and structural properties. Thus, MLCh-dependenthydrolysis of free MBP oligopeptides can to some extent be aconsequence of their partial homology and possible to similarity ofspatial properties.

Figure 5: Determination of the KM and kcat values for MBP (A) andthree OPs (B) in the reaction (pH 7.0) catalyzed by MLCh (30 nM)in the presence of 3 mM CaCl2 using the Lineweaver–Burk plot.The error in the initial rate determination from two independentexperiments at each substrate concentration did not exceed 7–10%.

Affinity of MLCh for substratesThe MLCh in the presence of 3 mM CaCl2 (pH 7.0) demonstrated

Km for intact MBP (15 ± 1.1 μM) and kcat value 0.4 ± 0.03 min-1

(Figure 5A). The Km and kcat values for three different OPs were alsodetermined: OP19 (0.5 ± 0.02 mM, 25.2 ± 1.2 min-1), OP21 (0.3 ± 0.02mM, 95.2 ± 6.2 min-1), and OP25 (0.2 ± 0.02 mM, 16.6 ± 1.2 min-1)(Figure 5B).

DNase activity of MLChTwenty four of 72 MLChs efficiently hydrolyze only MBP. Only one

of twenty five MLChs (NGTA3-pro-DNase) efficiently hydrolyzed notonly MBP, but also DNA (for example, Figure 6A). It was shown, thatonly NGTA3-pro-DNase of twenty five MLChs demonstrated positiveELISA answer using plates with immobilized DNA with mouse IgGs(conjugated with horseradish peroxidase) against human Abs lightchains.

To exclude possible artefacts due to hypothetical traces ofcontaminating canonical DNases, the purified preparation of NGTA3-pro-DNase was subjected to SDS-PAGE with following analysis ofDNase activity of eluates of the gel cross sections similarly to analysisof MBP-hydrolyzing activity. DNase activity was detected only in theband corresponding to the MLCh-25 (Figure 2A). I addition, DNase

activity of NGTA3-pro-DNase was revealed using in situ analysis afterseparation of proteins in gradient SDS-PAGE gels co-polymerized withcalf thymus DNA (Figure 6B).

Figure 6: DNase activity of three different MLChs (10 nM)including NGTA3-pro-DNase (lane MLCh1) in the cleavage ofscDNA was analysed in the presence of 5 mM MgCl2 (A); lane Ccorresponds to scDNA incubated alone. In situ assay of DNaseactivity of the NGTA3-pro-DNase (8 μg) after treatment with DTT(lane A) (B). DNase activity was revealed by ethidium bromidestaining as a dark band on the fluorescent background. A part of thegel was stained with Coomassie R250 to show the position of theSLE IgGs before (lane 1) and after incubation with DTT (lane 2), aswell as NGTA3-pro-DNase (lane 3). MLCh was analyzed byWestern blotting to a nitrocellulose membrane using mouse IgGsagainst light chains of human Abs conjugated with horsedishperoxidase (lane WB). The dependence of RA of MLCh on theconcentration of different metal ions (C). The dependence of RA ofMLCh on the concentration of NaCl or KCl in the presence of 5mM MgCl2 (D). The dependence of the relative DNase activity ofMLCh on pH of reaction mixture is given (E). Determination of theKM and kcat values for DNA (F) in the reaction (pH 6.5) catalyzedby MLCh (0.3 nM) in the presence of 5 mM MnCl2 and 30 mMNaCl using the Lineweaver–Burk plot. The error in the initial ratedetermination from two-three independent experiments in all casesdid not exceed 7–12%. For details, see Materials and Methods.

Ethidium bromide staining of the gels after the electrophoresis ofthe MLCh revealed sharp dark bands against a fluorescent background

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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of DNA in the gel zone corresponding only to the MLCh-25 and therewere no other peaks of proteins or DNase activity (Figure 6B). MLChdemonstrated positive answer at Western blotting to a nitrocellulosemembrane using mouse IgGs against light chains of human Absconjugated with horsedish peroxidase (Figure 6B).

Dependence of MLCh DNase activity on different metal ionsMany pro- and eukaryotic DNases including human DNase I (h-

DNase I) [40,41] as well as DNase abzymes from the sera of patientswith different pathologies [3-6,34] are Me2+-dependent, while otherDNases including DNase II and some subfractions of DNase sIgAsfrom human milk are Me2+-independent. Therefore, we have analyzedthe relative activities (RAs) of the MLCh before and after its dialysisagainst EDTA; after dialysis the MLCh completely lost the activity, butrecovered DNase activity after addition of different external metal ions(Figure 6C). It is known that optimal Me2+-cofactor of DNase I is Mg2+

and its optimal concentration is 10 mM [40,41]. Other metal ions veryweakly activate DNase I. At the same time, the maximal and nearly thesame activity at 2 mM MeCl2 was observed for Mn2+, Ca2+, and Mg2+

(Figure 6C). Optimal concentration for Mn2+, Mg2+, and Ni2+ wasobserved at their 5 mM concentration, while for Ca2+ and Zn2+ at 2mM. In addition, the increase in Ca2+ concentration higher 2 mM ledto the inhibition of MLCh-25 DNase activity (Figure 6C). Interestingly,the DNase activity of MLCh was increased in the presence of Co2+ andCu2 up to 10 mM concentration. In contrast to DNase I, Mn2+ andCo2+ are the best activators of MLCh-25. MLCh-25 demonstrate a veryindividual dependence of DNase activity on different metal ions (Mn2+

≈ Co2+ ≥ Mg2+>Cu2+ ≈ Ni2+ ≥ Ca2+>Zn2+), which is completelydifferent in comparison with that for DNases I.

Figure 7: Scatchard plot for oligonucleotide binding to MBP asmeasured by decrease in fluorescence emission.

It was shown recently, that the optimal concentrations of NaCl orKCl in the case of various MLChs having only DNase activity may bevery different and varied in range 10-100 mM [18,19]. Therefore, wehave compared the dependences of DNase activity of NGTA3-pro-DNase on concentrations of NaCl or KCl in the presence of Mg2+ andMn2+ at fixed concentration (5 mM). One can see that at 30-40 mM ofNaCl or KCl the activity of NGTA3-pro-DNase is higher for only10-15% (Figure 6D). Activation of DNases in the presence of Na+ and

K+ is usually associated with optimization of the structures of DNAand enzymes for effective catalysis.

pH optimum of DNase activity of MLChThe pH optima for various canonical DNases are very different, but

each of them usually demonstrates only one pH optimum [40,41]. Incontrast to all canonical DNases, pool of polyclonal DNase Abs fromthe sera patients with different diseases can contain from one to manymonoclonal abzymes; some preparations can demonstrate from one to2-8 pronounced optima at pH range from 5 to 10 [3-6,34]. We haveanalyzed pH optimum for the MLCh-25 in the presence of 5 mMMnCl2 and 30 mM NaCl. MLCh-25 demonstrated well expressed pHoptima at pH 6.5-6.6 (Figure 6E). Finally using optimal conditions theKm (2 ± 0.2 nM) for scDNA (and kcat= (1.1 ± 0.1) × 10-3 min-1) in thereaction catalyzed by MLCh-25 was determined (Figure 6F).

DiscussionAn extreme diversity of polyclonal IgG, IgA, and IgM abzymes in

their affinity for MBP and DNA was shown previously using differentmethods [9-12,16,17,42]. Interestingly, when polyclonal IgGs wereeluted from MBP-Sepharose or DNA-cellulose by a NaCl gradient (0-3M), the Ab optical density and proteolytic activity were distributed allover the chromatography profiles. Several fractions were eluted fromboth sorbents only with 2-3 M MgCl2 or with acidic buffer (pH 2.6) inthe conditions destroying strong immunocomplexes. We have expectedsimilar situation using chromatography on MBP-Sepharose in the caseof separation of phage particles containing kappa light chains on theirsurfaces. Figure 1A shows the distribution of the phage particles (andtheir MBP-hydrolyzing activity) all over the profile of thechromatography on MBP-Sepharose. The data are indicative of theextreme diversity of SLE anti-MBP recombinant kappa light chains intheir affinity for MBP. In previously published [35-37] and this articlefor a preparation of individual recombinant MLChs we have usedphage particles eluted from MBP-Sepharose with 0.5 M NaCl (Figure1A). One of 25 MLChs efficiently hydrolyzed not only MBP, but alsoDNA.

Many known canonical human metalloproteases are calcium-dependent cysteine proteases [50,51] or Zn2+-dependent enzymes [52],while their activity in the presence of other metal ions is absent orsignificantly lower. MLCh-25 is not cysteine protease demonstratingmaximal activity in the presence of Ca2+ and Ni2+, but effects of fiveother metal ions on its activity are to some extent comparable (Figure3B). In addition, serine-like activity of MLCh-25 is approximately 2.2-fold lower that its Me-dependent activity in the presence of Ca2+ Ni2+

ions.

All known canonical mammalian proteases have one pronouncedpH optimum. It was shown, that after the treatment of MLCh-25 withPMSF pH optimum of metalloprotease activity corresponds to 8.5(Figure 3E). After suppression of Me2+-dependent activity by EDTA,maximum of serine-like protease activity was observed at 7.0 (Figure3E) and both pHs were different in comparison with human trypsinand chymotrypsin (7.8-8.0).

It is known, that canonical proteases efficiently hydrolyze allproteins. After the separation on MBP-Sepharose polyclonal SLE andMS IgGs hydrolyze only MBP (see above). MLCh-25 hydrolyzesefficiently only MBP and three of four different OPs corresponding tofour different immunodominant regions of MBP (Figure 3F). Sincespecific inhibitors of different proteases except PMSF and EDTA did

Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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not decrease remarkably MBP-hydrolyzing activity of MLCh (Figure3A), it may be considered as unusual protease specifically recognizingand hydrolyzing only MBP due to two alternative active centers withserine-like and metalloprotease activities.

The affinity of polyclonal SLE and MS IgGs to MBP varied from 0.1to 1 μM [6,16,17], while affinity of MLCh-25 to MBP 10-100-foldlower (15 μM) (Figure 5A). In addition, affinity of three different OPs(0.2-0.5 mM) to MLCh is about 13-30-fold lower than that to MBP(Figure 5).

The catalysis mediated by artificial abzymes is usually characterizedby relatively lower reaction rates than for canonical enzymes [3-6,34].The known kcat values for natural abzymes from autoimmune patientscatalyzing many different chemical reactions vary in the range of 1 ×10-5-40 min–1([3-6,34,53] and refs therein). NGTA3-pro-DNasedemonstrated relatively high kcat value in the hydrolysis of OPs[16.6-95.2 min-1] and lower in the hydrolysis of OBM (0.4 min-1).Importantly, globular molecules of intact IgGs interact with variableparts of both heavy and light chains of abzymes [3-6,34]. At the sametime, the catalytic centers of abzymes, including proteolytic ones, areusually located on the light chain, while the heavy chain is mainlyresponsible for the specific antigen recognition and the increasedantigen affinity for abzymes. The separation of the light chains leads todecrease in the substrate affinity and in the lifetime of the existence ofthe complex and, as a consequence, to an increase in the turnovernumber and Vmax (kcat) of the reaction [3-6,34]. As it was shownpreviously, short oligopeptides interact mostly with the light chain ofintact Abs, which possesses a 100-1000-fold lower affinity for intactproteins [17-53]; similar situation was observed for MLCh-25. Higheraffinity and lower kcat for MLCh-25 in the case of MBP may be due tosome specific additional contact of light chain with MBP incomparison with OPs.

It is obviously, that MLCh-25 should correspond to Abs against onlyone antigenic determinant of MBP. At the same time, it efficientlyhydrolyzes three of four OPs corresponding to MBP (Figure 3F). It wasshown that the hydrolysis of nonspecific OPs corresponding to MBP byanti-integrase abzymes of HIV infected patients is caused by a partialhomology between sequences of oligopeptides and several fragmentsof integrase [54]. Figure 4B demonstrates that four different OPs ofMBP are to some extent homologous. Since OP21 is the best substrateone can propose that MLCh correspond to Abs against this sequenceof MBP. At the same time, MLCh efficiently hydrolyzes only three offour OPs (Figure 3F). Probably, that not only partial homology, butalso spatial structures of free OPs of the same oligopeptides withinglobular proteins can play important role in their cleavage by abzymesagainst MBP. One cannot, that in spite of comparable homologybetween four OPs (Figure 4B), spatial structure of free OP17 may benot optimal for cleavage in comparison with that for free OP21.

The most unexpected result is that in addition to the two types ofproteolytic activity, MLCh-25 also possesses DNase activity. It wasshown that DNase activity is intrinsic property of NGTA3-pro-DNase(Figure 6A). The Km and kcat values for polyclonal Abs against DNAare usually varied in the ranges 0.5-10 nM and 10-5–40 min-1,respectively [3-6,34,53]. The affinity of MLCh for scDNA turned out tobe unexpectedly high, Km=2.0 ± 0.2 nM, and as a consequence the kcatwas relatively low (1.1 ± 0.1) × 10-3 min-1. The affinity of scDNA forMLCh-25 (in terms of KM values) is about 3.5 orders of magnitudehigher than affinity of scDNA for DNase I (KM=46-58 μM) [53], whilekcat is significantly lower (kcat for DNase I is approximately 1.0 × 105

min-1). The question is why MLCh-25 against MBP can hydrolyze

DNA. It is believed that MBP and anti-MBP Abs cannot interact withDNA or RNA. It was recently shown that anti-MBP Abs efficientlyinteract with nucleic acids [55]. It should be noted, that MBP is a veryhydrophobic protein. Therefore, we did not exclude, that MBP can alsointeract with different molecules including nucleic acid. Quenching ofMBP tryptophan fluorescence emission decrease was used for theestimation of the MBP affinity for oligonucleotide. Figure 7demonstrates Scatchard plot of the dependence of relative decrease influorescence on the concentration of oligonucleotide. Interaction ofpure MBP with oligonucleotide is characterized by two Kd values: 65 ±5 and 250 ± 20 μM. It means that MBP in principle can interact withDNA. In addition, one cannot exclude that the affinity of MBP as acomponent of axons myelin can be higher due to interaction of DNAwith other components of myelin (different proteins, oligosaccharides,and lipids) contacting with MBP.

It is possible to suggest that 24 of 25 MLCh interacting only withMBP correspond to Abs directly against this protein [35-37], whileNGTA3-pro-DNase may be against the complex of MBP with DNA. Inthe latter case, it is impossible to exclude possibility of a formation ofthe chimeric MLChs possessing affinity for MBP and for DNA, andalso hydrolyzing these absolutely different substrates.

Three different activities of MLCh-25 were extremely unpredictable,but this phenomenon may be important for the further development instudies of the yet unknown possible biological functions ofimmunoglobulins.

AcknowledgmentsThis research was made possible in part by grants from the

Presidium of the Russian Academy of Sciences (Molecular and CellularBiology Program, 6.2), Russian Foundation for Basic Research(13-04-00208; 13-04-00205), and funds from the Siberian Division ofthe Russian Academy of Sciences. We are very grateful S. Paul and S.Plaque (University of Texas Houston Medical School, USA) forgranting to us cDNA library of SLE patients.

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Citation: Timofeeva AM, Buneva VN, Nevinsky GA (2017) SLE: Unusual Recombinant Monoclonal Light Chain NGTA3-Pro-DNase PossessingThree Different Activities Trypsin-like, Metalloprotease and DNase. Lupus Open Access 2: 127.

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Lupus Open Access, an open access journal Volume 2 • Issue 2 • 127